1. Field Of The Invention
This invention relates to a non-A, non-B hepatitis virus antigen peptide. More particularly, the present invention is concerned with a non-A, non-B hepatitis virus antigen peptide which exhibits antigen-antibody reaction specificity with at least one of a convalescent serum from a patient having acute non-A, non-B hepatitis and a serum from a patient having chronic non-A, non-B hepatitis. The antigen peptide of the present invention is useful for diagnosing non-A, non-B hepatitis and for screening blood for transfusion. Further, the antigen peptide of the present invention can be used as an active ingredient of a vaccine for non-A, non-B hepatitis.
2. Discussion Of Related Art
Definition of non-A, non-B hepatitis virus:
The viral hepatitis is a liver disease caused by the infection of a hepatitis virus. Heretofore, hepatitis A virus, hepatitis B virus and hepatitis D (delta) virus have been isolated and identified. The hepatitis D virus (delta-hepatitis virus) is a deficient virus which cannot grow by itself and requires for its growth the co-presence of hepatitis B virus as a helper virus. Therefore, the hepatitis D virus is present only in a patient having hepatitis B. In 1974, it was reported that there were many patients having hepatitis caused by a factor other than the infection with either hepatitis A virus or hepatitis B virus. Such a hepatitis was named "non-A, non-B hepatitis", and researches on the non-A, non-B hepatitis virus have been made extensively and intensively throughout the world. Heretofore, it has been found that a plurality of types of non-A, non-B hepatitis viruses exist. Results of the researches up to now show that the non-A, non-B hepatitis virus is classified into two types according to the infection route, that is, an epidemic hepatitis virus, namely an enterically-transmitted non-A, non-B hepatitis virus, which is spread through water and foods; and a blood-transmitted non-A, non-B hepatitis virus which is spread through blood by transfusion, etc. Of the non-A, non-B hepatitis viruses, only an enterically-transmitted NANBV which spreads over the areas of Africa, India and Southeast Asia has been virologically identified, but the blood-transmitted non-A, non-B hepatitis virus has not yet been identified.
Hereinbelow, the blood-transmitted non-A, non-B hepatitis is often referred to simply as "NANB hepatitis", and the blood-transmitted non-A, non-B hepatitis virus is often referred to simply as "NANBV". Current situation of the studies on NANB hepatitis and Problems:
With respect to the mechanism of the crisis of the NANB hepatitis and the treatment of the NANB hepatitis, virological studies have been made in the world by the comparison of NANBV with the other hepatitis viruses, diagnostic, histopathology and the like (Japan Medical Journal, No. 3220, pp. 3-10, 1987). With respect to the NANB hepatitis, the following findings have been reported.
(1) Epidemiology: In Japan, according to the estimation by Ministry of Health and Welfare, about 60% of chronic hepatitis patients (namely about 720 thousand patients), about 40% of hepatocirrhosis patients (namely about 100 thousand patients) and about 40% of liver cancer patients (namely about 7 thousand patients) are patients having NANB hepatitis. Further, the motality rate attributed to the above-mentioned NANB hepatitis reaches 16 thousand per year. In U.S.A., the number of post-transfusion hepatitis patients reaches 150 to 300 thousand per year and 90% of the post-transfusion hepatitis patients are patients having NANB hepatitis. Further, it is considered that 1 to 6% of the blood donors are an NANBV carrier. Further, it is estimated that in the other countries also, the incidence of NANB hepatitis and the ratio of the NANBV carrier are equal to or higher than those in U.S.A. and Japan. Therefore, prevention, early diagnosis and early treatment of the NANB hepatitis is a matter of global importance.
(2) Virology: The NANBV heretofore reported comprises an envelope and a viral particle having a spherical shape of a diameter of about 50 nm. The taxonomic observation suggests that the known NANBV is a virus similar to a togavirus or a flavivirus, or a virus of new type different from the togavirus or the flavivirus. Further, the results of pathological observations of the cytoplasms of hepatocytes of a plurality of chimpanzees injected with a blood serum of a patient having NANBV hepatitis show that the formation of a tubular structure is observed in the cytoplasm of a hepatocyte of some of the chimpanzees, but not observed in the cytoplasm of a hepatocyte of the other chimpanzees, and that an intranuclear particle is formed in the cytoplasm of a hepatocyte of some of the chimpanzees. These results and the results of the epidemiological observation and the tests on the presence or absence of the chloroform sensitivity suggest that a plurality of types of NANBV's exist, (see, for example, "Science", Vol. 205, pp. 197-200, 1979, and "Journal of Infectious Disease", Vol. 148, pp. 254-265, 1983). The amount of the NANBV present in the blood of a patient having NANB hepatitis is extremely small as compared to either the amount of a hepatitis A virus present in the feces of a patient having hepatitis A or the amount of a hepatitis B virus present in the blood of a patient having hepatitis B. For example, the amount of hepatitis B virus in the blood of the patient is 10.sup.8 to 10.sup.9 per ml in terms of Chimpanzee Infectious dose (CID), whereas the amount of NANBV in the blood of the patient is only 10.sup.4 to 10.sup.5 per ml in terms of CID (Bradley, D.W.: Research perspectives in post-transfusion non-A, non-B hepatitis, in "Infection, Immunity and Blood Transfusion", edited by Dodd, R.Y. & Barker, L.F., published by Alan R. Liss, Inc., New York (1985) pp. 81-97). Further, it is known that except for human, there are no animals except chimpanzee that are sensitive to NANBV and that in the cytoplasm of the hepatocyte, a typical tubular structure is occasionally formed by NANBV infection. Since only chimpanzee can be used as an animal for experiment of the NANBV infection, a large number of chimpanzees are required to be used for the study of NANBV. However, the chimpanzee is not easily available and expensive. Therefore, the study of NANBV by, for example, experimental infection by NANBV, identification of NANBV and search for a useful marker for NANBV, is necessarily restricted and delayed.
(3) Clinical observation: The latent period of the NANB hepatitis is 2 to 26 weeks. The symptom of NANB hepatitis in the early stage is mild as compared to that of hepatitis B. For example, a patient having NANB hepatitis only becomes feverish and complains of languor. Further, 70% of the patients have anicteric symptom. Therefore, the NANB hepatitis is frequently overlooked. However, the NANB hepatitis is very dangerous because the NANB hepatitis is likely to become chronic and, then, to progress to liver cirrhosis. That is, 40 to 50% of the patients having NANB hepatitis whose blood serum exhibits an increased aminotransferase activity develop chronic hepatitis. 10 to 20% of the cases of chronic hepatitis are accounted for by liver cirrhosis. Further, 0.5 to 1% of blood recipients per year become liver cirrhosis patients without subjective symptoms. Therefore, for preventing biohazard caused by blood transfusion and bleeding, eradication of the NANB hepatitis is a matter of global importance from the viewpoint of public health.
(4) Diagnosis: As mentioned above, the NANBV (blood-transmitted type) has not yet been identified and a viral marker, such as an NANBV antigen, useful for the diagnosis of NANB hepatitis has not been known. Therefore, diagnosis of NANB hepatitis has been conducted by examining the titer of the antibody in serum of a patient, which is specific for each of the known phlogogenic viruses, such as hepatitis A virus, hepatitis B virus, cytomegalovirus, EB virus, varicella virus ad herpes simplex virus, and diagnosing the patient whose serum is negative with respect to the antibody specific for any of the above-mentioned viruses, as having NANB hepatitis. At the same time, another diagnosis method has also been used. For example, there have been used a method in which whether or not the activity of an enzyme in serum, such as GPT [glutamic-pyruvic transaminase, also known as "ALT" (alanine aminotransaminase)], GOT (glutamic-oxaloacetic transaminase, also known as "AST" (aspartate aminotransaminase)], and guanine deaminase (also known as "guanase") ("Kan-tan-sui (Liver, Gallbladder, Pancreas)", Vol. 14, pp. 519-522, 1987); a method in which guanase activity is determined based on the absorbance of a sample (Japanese Patent Application Laid-Open Specification No. 60-176600); and a method in which the activity of reverse transcriptase in blood is determined (U.S. Pat. No. 4,707,439). With respect to the GPT or GOT in serum mentioned above, standard for the diagnosis of NANB hepatitis in which lasting, abnormally high activities of GPT and GOT are utilized as a criterion for the diagnosis of NANB hepatitis, is employed in Japan ("Journal of Blood Transfusion Society in Japan", Vol. 31, No. 4, pp. 316-320, 1985). With respect to the diagnostic agent for NANB hepatitis, an antibody specifically reactive with an antigen which is formed in the blood of a patient having the symptoms of the NANB hepatitis is known. For example, a polyclonal antibody (Japanese Patent Application Laid-Open Specification No. 58 -735), a monoclonal antibody (Japanese Patent Application Laid-Open specifications No. 58-183629, No. 61-56196 and No. 62-181798), a monoclonal antibody produced by a cell strain (Japanese Patent Application Laid-Open Specification No. 61-25484), an avidin-labeled antibody (Japanese Patent Application Laid-Open Specification No. 60-195454), and an antibody prepared from the serum of a monkey infected with an extract of a stool of a patient having NANB hepatitis (Japanese Patent Application Laid-Open Specification No. 62-249999) are known. Further, an NANB hepatitis-related antigen which is isolated and purified from anatomized liver of a patient having NANB hepatitis (Japanese Patent Application Laid-Open Specification No. 57-198867) has been reported. However, the diagnosis of NANB hepatitis has not been able to be attained accurately by the use of any of the above-mentioned methods, antibodies and antigen, because the enzymatic activity utilized as a criterion for the diagnosis of NANB hepatitis in the above-mentioned methods is not specific for the NANB hepatitis, and the antibodies and the antigen mentioned above are not specific for NANBV. Therefore, it has been desired to obtain a marker specific for NANBV.
(5) Therapy and Prevention: Recently, the therapeutic effect on chronic NANB hepatitis by the use of interferon has been attracting attention in the art ["Hepatology", Vol. 6, p. 1117, 1986 (abstract)]. With respect to the dose of .beta.-interferon and the period of administration, investigations have been made. However, the effectiveness of .beta.-interferon has not yet been confirmed.
On the other hand, the various vaccines and diagnositic agents have been reported. For example, there can be mentioned an adjuvant vaccine containing an antigen derived from a serum of a patient having NANB hepatitis who has a symptom of a jaundice, which serum exhibits high GPT activity (Japanese Patent Application Laid-Open Specification No. 59-42455); an NANBV antigen specifically reactive with an antibody produced by a lymphocyte obtained from a patient having NANB hepatitis which lymphocyte has been transformed with EB virus which has been isolated and purified from cells of the liver of a chimpanzee having NANB hepatitis (Japanese Patent Application Laid-Open Specification No. 61-176856); a glycoprotein derived from a blood serum of a patient having NANB hepatitis (Japanese Patent Application Laid-Open Specification No. 62-30965); a DNA virus isolated from a blood serum of a patient having NANB hepatitis by affinity chromatography (Japanese Translation Publication No. 59-501774 of PCT Application Publication No. WO84/01107); an NANBV antigen isolated from the fibronectin fraction of blood plasma (Japanese Translation Publication No. 60-501241 of PCT Application Publication No. WO84/04326); a togavirus isolated from a serum and an urine of a patient having NANB hepatitis (U.S. Pat. No. 4,464,474); a vaccine containing, as an active ingredient, an NANBV surface antigen or a gamma globulin purified from a serum or a plasma of a patient having NANB hepatitis (U.S. Pat. No. 4,542,016); an NANBV purified from a serum of a patient having NANB hepatitis, and an antigen thereof (U.S. Pat. Nos. 4,673,634 and 4,702,909 and European Patent Application Publication No. 128,995); and an enterovirus-like virus isolated from an extract of a stool of a patient having NANB hepatitis (European Patent No. 71,640). However, since the NANBV have not yet been isolated, any confirmation cannot be made as to whether or not the above-mentioned vaccines and diagnostic agents are surely useful for specifically diagnosing and treating NANB hepatitis. Furthermore, with respect to the method for preventing an NANBV from spreading via blood preparation, there have been known, for example, a method in which a serum or a blood plasma is subjected to heat-treatment for 10 min at 60.degree. C. (U.S. Pat. No. 4,438,098); a method in which freeze-dried blood preparations are prepared and subjected to heat-treatment (Japanese Patent Application Laid-Open Specification No. 59-110627); a method in which a fraction of a plasma protein precipitated from an organic solvent, such as chloroform, is collected and the organic solvent-soluble fraction containing the hepatitis viruses are discarded; and a method for the inactivation of NANBV by formalin (U.S. Pat. No. 4,291,020). However, as mentioned above, the NANBV has not yet been isolated and identified and, therefore, any confirmation cannot be made as to whether or not the blood treated by the above-mentioned methods is surely free from the NANBV. Therefore, the safety and effectiveness of the biological preparations obtained by the above-mentioned method are not insured. Accordingly, none of these methods have been put into practice use.